The invention relates to methods for specifically inactivating myelin proteins which inhibit nerve regeneration. These methods are useful to promote axon regeneration.
Axon regeneration occurs from a severed end by forming a new neuronal growth cone, the sensory motile organelle at the ends of axons that is responsible for neurite extension and axon guidance of developing neurons. While central nervous system (xe2x80x9cCNSxe2x80x9d) neurons do not regenerate after injury, peripheral nerves do. For example, severed nerves within the adult optic nerve generally do not regenerate (reviewed in Aguayo et al., 1991). It is thought that if the severed nerves reform circuitry, then significant function can be reestablished (Cheng et al., 1996).
David et al. (1981) showed that CNS nerves extended axons into bridges of peripheral tissue demonstrating that the difference between the CNS and peripheral nervous system environments is critical for regeneration. It has recently been shown that the major difference in regenerative capacity is due to inhibitory molecules present in the myelin sheaths that surround spinal cord axons of the adult CNS (Schwab et al., 1993). Spinal cord neurons show increased collateral sprouting when myelin is absent (Schwegler et al., 1995). Application of a myelin-derived fraction to neurons cultured in the absence of myelin caused growth cone collapse and neurite retraction (Caroni and Schwab, 1988a).
It has been demonstrated that at least one inhibitory property of myelin extracts can be partially neutralized by an antibody called IN-1 (Caroni and Schwab 1988b). The application of this antibody to injured rat spinal cord can promote a small amount of axon regeneration (Schnell and Schwab, 1990; Bregman et al., 1993). IN-1 also caused a small amount of regeneration after optic nerve lesion (Weibel et al., 1994). Regeneration was incomplete in these reported studies and in no case was reconnection made to former targets. This may have been due in part to the fact that IN-1 is only partially blocking (the antigen has yet to be purified). Also, there are several other inhibitory molecules present in the myelin, some that have yet to be identified (Schwab et al., 1993). Identifying these other molecules in myelin that inhibit regeneration would aid in developing treatment for CNS nerve injury.
Another neurite inhibitory protein derived from myelin (distinct from the IN-1 antigen) is the abundant myelin-associated glycoprotein (MAG) (McKerracher et al., 1994). Purified MAG can inhibit neurite outgrowth from peripheral sensory (DRG) neurons and neuroblastoma-derived cell lines such as NG-108 cells (McKerracher et al., 1994; Mukhopadhyay et al. 1994). Immunodepletion of myelin using anti-MAG antibodies removed 63% of its neurite growth inhibition. Together, these findings argue that MAG may inhibit regeneration after injury but this remains controversial. (Bartsch et al. 1995; Schafer et al. 1995) Establishing whether this abundant myelin protein is inhibitory for regeneration of retinal axons would be significant. If so, devising strategies to selectively destroy its activity would provide novel therapies after optic nerve or spinal cord injury.
It is an object of this invention to provide methods that inactivate the inhibitory proteins within myelin and thus promote axon regrowth and regeneration of CNS nerves. It is another object of this invention to provide a method of specifically inactivating myelin-associated glycoprotein and thus promote axon regrowth and regeneration.
The inventors have discovered that the inactivation of myelin associated glycoprotein (MAG) promotes axon regrowth and regeneration in damaged CNS neurons. In one embodiment, the invention is a method of promoting axon regeneration of a CNS nerve in the tissue of a mammal by specifically inactivating the function of MAG using chromophore-assisted laser inactivation (CALI).
The method of treatment is conducted in a patient as follows, using optic nerve as an example. A damaged optic nerve of a patient is contacted with an anti-MAG antibody having a malachite green chromophore tag, for example by infusing the antibody into the eye or eye cavity using a catheter for a time and under conditions sufficient to allow binding of the antibody to MAG in the myelin of the optic nerve. The optic nerve is then irradiated with a laser beam at a wavelength of 620 nm for a time sufficient to activate the malachite green chromophore tag which tag produces short-lived free radicals that selectively and irreversibly inactivate the function of the MAG bound to the antibody without damage to the tissues of the patient. The turnover time for MAG in myelin is approximately 40 days, thus additional treatment may be required to selectively inactivate the MAG in the myelin of the optic nerve until a time at which axon regeneration is complete.
In a preferred embodiment the method of the invention is directed to axon regeneration any of the following CNS nerves, any of the twelve cranial nerves (such as the optic nerve and auditory nerve), spinal cord and nerves which carry signals into the spinal cord (e.g. sciatic nerve).